Synthesis and evaluation of stimulatory properties ofSphingomonadaceae glycolipidsXiangtian Long1,5, Shenglou Deng1,5, Jochen Mattner2, Zhuo Zang1, Dapeng Zhou3, Nathan McNary1,Randal D Goff1, Luc Teyton4, Albert Bendelac2 & Paul B Savage1

Glycosphingolipids (GSLs) from the Sphingomonadaceae familyof bacteria have been reported to be potent stimulators of naturalkiller T cells. These glycolipids include mono-, tri- andtetraglycosylceramides. Here we have prepared the GSL-1 toGSL-4 series of glycolipids and tested their abilities to stimulatenatural killer T cells. Among these glycolipids, only GSL-1 (1) is apotent stimulator. Using a series of synthetic diglycosylceramides,we show that oligoglycosylceramides from Sphingomonadaceaeare not effectively truncated to GSL-1 in lysosomes in antigen-presenting cells, possibly because the higher-order GSLs are poorsubstrates for lysosomal acyltransfer enzymes.

The innate immune system is central to controlling microbial growth,and a key aspect of this system is continuous surveillance forcompounds that indicate the presence of microbes. For example,recognition and inflammatory responses to lipid A (2) are mediatedthrough specific binding of this glycolipid by the receptors CD14 andTLR4 on monocytes, macrophages and other antigen-presenting cells(APCs)1. Although many well-studied organisms produce lipid A,several Gram-negative bacteria do not secrete this glycolipid2–4, raisingthe question: does the innate immune system survey for the presenceof glycolipids from these bacteria? Among Gram-negative bacteria thatdo not produce lipid A, the best-studied outer membrane components

O

OHOHOO

CO2H

C14H29

HN

OH

O

H2NHO

HOO

O

OHHO

HO

HO

C13H27

C14H29

C25H51

C13H27

C13H27

OH

O

O

OHOHO

HOHO2C

C14H29

HN

OH

C13H27

OH

O

GSL-1

GSL-3 GSL-4

O

OHOHOO

CO2H

HN

OH

O

H2NHO

HO

O

O

OHHO

HO

OH

O

O

OH

HOHO

HO

O

O

OHO

HO

HOOH

(CH2)13

(CH2)13 (CH2)7CH3

HN

OH

C22H45

C25H51

C25H51

C14H29 C14H29

C25H51

C14H29

C25H51

C14H29

C25H51C14H29

C25H51

C14H29 C14H29

O

OH

OH

O

OO

HO

HOOH

HN

OH

O

OH

O

HO

HOHO

OH

O

OHO

HO

OOH

HN

OH

O

OH

O

AcHNHO

HOOH

O

OHO

HO

OOH

HN

OH

O

OH

O

OHHO

HO

HO

α-Gal-(1-2)-α-GalCer

Glycolipid isolated from Agelas mauritianus

O

OHO

HO

HOOH

HN

OH

O

OH

KRN7000 (α-GalCer)

α-Gal-(1-4)-α-GalCer β-GalNAc-(1-4)-α-GalCer α-GlcNH2-(1-4)-α-GalCer

O

OHO

HO

OOH

HN

OH

O

OH

O

H2NHO

HO

OH

α-GlcNAc-(1-4)-α-GalCer

O

OHO

HO

OOH

HN

OH

O

OH

O

AcHNHO

HO

OH

O

OHOHO

OCO2H

C14H29

HN

OH

O

H2NHO

HOHO

C13H27

OH

O

'GSL-2'

O

OHO

HO

HONHAc

HN

OH

O

OH

O O

HOOHO

HN

OH

O

iGb3

OH

O

OH

O

OHOHO

HO

HO

HOOH

PBS57

Figure 1 Structures of potential NKT-cell agonists. Shown are structures of glycolipids from the Sphingomonadaceae family of bacteria, an

a-galactosylceramide isolated from the sponge A. mauritianus, the synthetic NKT cell agonists KRN7000 and PBS57, endogenous antigen iGb3, and

diglycosylceramides used to observe lysosomal processing of glycolipids.

Received 7 May; accepted 30 June; published online 29 July 2007; doi:10.1038/nchembio.2007.19

1Department of Chemistry and Biochemistry, C100 BNSN, Brigham Young University, Provo, Utah 84602, USA. 2Department of Pathology, 57 East 7th Street,University of Chicago, Chicago, Illinois 60637, USA. 3Department of Melanoma Medical Oncology, University of Texas M.D. Anderson Cancer Center, 7455 FanninStreet, Houston, Texas 77054, USA. 4Immunology Department, 10550 North Torrey Pines Road, Scripps Research Institute, La Jolla, California 92037, USA. 5Theseauthors contributed equally to this work. Correspondence should be addressed to P.B.S. (paul_savage@byu.edu).

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007 559

L E T T ERS

are a-glycosylceramides from the Sphingomonadaceae family of bac-teria2,5. This family includes bacteria to which humans are commonlyexposed, and human biliary cirrhosis has been correlated to infectionswith these organisms6.

Structures of glycolipids from Sphingomonas spp. have been pro-posed on the basis of spectroscopic measurements2. GSL-1 is amonoglycosylceramide, and GSL-3 (3) and GSL-4 (4) both incorporateglucosamine along with one or two additional sugars (Fig. 1). Most ofthese GSLs use C18 sphinganine, but a few of them incorporate C21

sphinganine containing a cis double bond or cyclopropyl group.We7, along with other groups of researchers8,9, have shown that

heat-killed Sphingomonas spp. and GSL-1 from Sphingomonas spp. arepotent stimulators of natural killer T (NKT) cells. In addition, it hasbeen reported NKT cells are stimulated by GSL-4 isolated fromSphingomonas9. To establish that bacteria outside the Sphingomona-daceae family are also active, we showed that NKT cell stimulatingproperties extend to other non-lipid A–producing bacteria in theAlphaproteobacteria subclass7.

NKT cells are regulatory T cells with a restricted repertoire of T cellreceptors, and consequently this cell type is thought to be ‘prepro-grammed’ to recognize a limited set of antigens10. Thus, NKT cellshave been classified as part of the innate immune system. Stimulationof NKT cells through presentation of glycolipids by the CD1d proteinon APCs results in a release of cytokines that influence responses ofother aspects of the immune system, and NKT cells have beenimplicated in responses to numerous disease states11,12. The consider-able impact of NKT cells on immune responses has prompted effortsto understand the types of glycolipid antigen that can stimulate thesecells. Much of the initial work with NKT cells focused on antitumorresponses to a glycolipid, KRN7000 (5), derived from a naturalproduct isolated from the marine sponge Agelas mauritianus13 (foran example, (6), see structure in Fig. 1). ‘Natural’ antigens for NKTcells have since been described7–9,14–16, and include both endogenousand exogenous glycolipids.

Many variants of KRN7000 have been prepared and tested for NKTcell stimulatory activity17. From these studies, influences of structuralvariation on CD1d presentation and NKT cell stimulation have beendetermined. For example, sugars appended on KRN7000 can influenceCD1d presentation and NKT cell stimulation: diglycosylceramides,including a-Gal-(1-2)-a-GalCer (7), a-Gal-(1-4)-a-GalCer (8) andb-GalNAc-(1-4)-a-GalCer (9; Fig. 1), stimulate NKT cells only if they

are truncated by glycosidases to give KRN7000 (refs. 15,18). Typically,glycolipids are transported to lysosomes in APCs, where they areexposed to a collection of glycosidases. a-Glycosylceramides withsmall molecules appended at C6¢¢ are tolerated by CD1d and thecorresponding T cell receptor and do not need to be truncated toallow stimulation of NKT cells19.

The structural similarities of KNR7000 and GSL-1 suggest thatstructural variations should have comparable effects on NKT cellstimulation, and it would be expected that GSL-3 and GSL-4 wouldhave to be truncated to GSL-1 to stimulate NKT cells. To determinewhether GSL-3 and GSL-4 are stimulatory, and to avoid possiblecontamination of these glycolipids with GSL-1 isolated from Sphingo-monas, we have synthesized these three glycolipids, compared theirstructures to those of isolated glycolipids, and determined their abilityto stimulate cytokine release from NKT cells. This effort has con-firmed the proposed structures of GSL-3 and GSL-4. We have alsoprepared ‘GSL-2’ (10) (Fig. 1) to determine the extent to which thecarbohydrates of the GSLs are recognized by NKT cells.

Of critical importance in understanding responses to GSLs is deter-mining the ability of glycosidases in the lysosome to process (that is,truncate) GSL-3 and GSL-4 to GSL-1. We therefore prepared theGSL-KNR7000 hybrids a-GlcNH2-(1-4)-a-GalCer (11) and a-GlcNAc-(1-4)-a-GalCer (12) (Fig. 1) to probe both for lysomal glycosidases thatcan remove a-glucosamine in the context of a-GalCer and for acyl-transfer enzymes that can acylate the amine in the former glycolipid.

To prepare the GSLs, we used a procedure that allowed the generationof all three GSLs found in Sphingomonadaceae and GSL-2, with anemphasis on convergent synthesis. The synthesis of GSL-1 hasbeen reported8, and synthesis of the higher-order GSLs required botha consideration of convergence and efficient manipulation of theprotecting group. The synthesis was based on known carbohydratecoupling procedures20–22; however, glucuronic acid is a poor glycosyldonor and acceptor, and its reactivity influenced the synthesis of allof the GSLs. As in all syntheses of oligosaccharides, control andverification of anomeric stereochemistry were central in the design ofthe synthesis. To maximize the convergence of the synthesis of GSL-4and to ensure that the anomeric configurations were correctly installed,appropriately protected GSL-2 was prepared and then the distaldisaccharide was incorporated. This final coupling involved a primaryalcohol and provided reasonable yields and stereocontrol late inthe synthesis.

OHO

N3

BnOBnO

AcO

SPhO

OH

PMBOBnO

BnO

SPhO

MeO2CPMBO

BnOBnO

SPhOTBSO

BnOBnO

HO+

+

C13H27

N3

OBn

O

OBnOBnO

TBSOMeO2C

C13H27

N3

OBn

O

OBnOBnO

HOMeO2C

C13H27

HN

OBn

C12H25

OPiv

O

O

OBnOBnOO

CO2Me

C13H27

HN

OBn

C12H25

OPiv

O

O

N3

BnOBnO

AcO

GSL-1 GSL-2

13 14 15 16 17

1820

a b c

d,e g

f h

19

MeO2C

Scheme 1 Preparation of GSL-1 and GSL-2. The reagents were as follows (yields in parentheses). (a) BIAB, TEMPO, CH2Cl2, H2O; CH2N2, Et2O (43%).

(b) DDQ, CH2Cl2; H2O, TBSCl, imidazole, DMF (81%). (c) Dimethyl(methylthio)sulfonium triflate, DTBMP, 4 A MS, CH2Cl2 (mixture of anomers; 67%).

(d) H2S, pyridine, S-2-hydroxytetradecanoyl chloride, pyridine (20% of the a anomer). (e) HF, CH3CN, H2O (87%). (f) MeONa, H2O, THF; Pd/C, H2, THF,

MeOH (40%). (g) Diphenylsulfoxide, Tf2O, tri-t-butylpyrimidine, 3 A MS, CH2Cl2 (70%). (h) MeONa, H2O, THF; Pd/C, H2, THF, MeOH (22%). Ac, acetyl;

Bn, benzyl; PMB, p-methoxybenzyl; TBS, t-butyldimethylsilyl.

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560 VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY

Preparation of GSL-1 and GSL-2 is shown in Scheme 1. Glucose witha p-methoxybenzyl group at C4 was oxidized to the glucuronic acidand converted to the methyl ester, giving 14. We found that at-butyldimethylsilyl ether at C4 did not completely withstand theoxidation conditions and that a p-methoxybenzyl ether complicatedlater steps in the synthesis. Consequently, it was necessary to exchangethe C4 protecting groups at this stage to give 15. Coupling with theazido version of sphingosine (16) gave a mixture of anomers, and onlyafter the azide had been reduced and coupled with S-2-hydroxyte-tradecanoic acid (giving 18) were the anomers separated. The poorglycosyl donor properties of 15 made it necessary to use azide 16,which is a better acceptor than the preformed ceramide. Deprotectionof 18 gave GSL-1. Coupling of 18 with 19 gave the a anomer in goodyield, and subsequent deprotection gave GSL-2.

The synthesis of GSL-3 followed a pathway similar to that used inthe synthesis of GSL-4 (see Supplementary Scheme 1 online). The keyconvergent step in the synthesis of GSL-4 (Scheme 2) was the couplingof 23 and 27. It was necessary to reduce the azide selectively and toprotect the resulting amine to avoid material losses late in thesynthesis, and we found that coupling of mannose with 25 gave thebest anomeric selectivity with perbenzoylated mannose. The benzoyl

groups tended, however, to slow the subsequent glycosylation. Wetherefore exchanged the ester groups on 26 to give 27 before the nextcoupling. The key convergent step occurred with good yield andanomeric selectivity (only a trace of the b-anomer was detected). Tofacilitate removal of the b-anomer, after reductive deprotection thematerial was peracylated to give 28, which was amenable to chroma-tography. Hydrolysis of the ester was followed by deprotection of theamine, producing GSL-4 in good yield.

GSL-4 can be readily isolated from Sphingomonas paucimobilis2, andwe directly compared isolated material to synthetic GSL-4. As notedabove, the structure of GSL-4 has been investigated spectroscopically2,and synthesis of the proposed structure and favorable comparison toisolated material provided additional evidence in favor of this proposedstructure. A challenge associated with the isolation of GSL-4 is thatGSL-1 is present and difficult to separate completely from GSL-4.Nevertheless, after careful chromatography, we isolated GSL-4 in pureform. The isolated material contained primarily the ceramide shown inFigure 1. Also present, however, was a GSL-4–containing ceramidederived from a longer-chained (C21) sphinganine with a double bond.Resonances from this double bond appeared as minor resonances inthe proton NMR spectrum of isolated GSL-4. Neglecting these minor

O

OO

Ph

BnOOBn

OHHO

+

C14H29

HN OAc

C25H51O

OAc

aO

OO

Ph

BnOBnO

OC14H29

HN OAc

C25H51O

OAc

O

OOTBS

BnO

BnOO

C14H29

HN OAc

C25H51O

OAc

O

OAc

BnOBnO

N3b

O

HOOTBS

BnO

BnOO

C14H29

HN OAc

C25H51O

OAc

c

d,e α-GlcNH2-(1,4)-α-GalCer

α-GlcNAc-(1,4)-α-GalCer

f

29 30 31 3233

Scheme 3 Preparation of GSL-KRN7000 hybrids a-GlcNH2-(1-4)-a-GalCer and a-GlcNAc-(1-4)-a-GalCer. The reagents were as follows (yields in

parentheses). (a) Diphenylsulfoxide, Tf2O, TTBP, 3 A MS, CH2Cl2 (62%). (b) TsOH, MeOH, CH2Cl2; TBSCl, imidazole, CH3CN (79%). (c) Diphenylsulfoxide,

Tf2O, TTBP, 3 A MS, 19, CH2Cl2 (51%). (d) HF, pyridine, THF (86%). (e) Na1, NH3, THF (56%). (f) Ac2O, DMAP, pyridine; MeONa, MeOH, THF (48%).

Ac, acetyl; Bn, benzyl; Ph, phenyl; TBS, t-butyldimethylsilyl.

SPhO

OBnBnO

BnO

OH

OO CCl3

NH

OBz

BzOBzO

BzO SPhO

OBnBnO

BnO

O

OBz

BzOBzO

BzO

O

SPhO

OBnBnO

BnO

O

OAc

AcOAcO

AcO

O

O

OBnOBnOO

CO2Me

C13H27

HN

OBn

C12H25

OPiv

O

O

N3

BnOBnO

TBDPSO

O

OBnOBnOO

CO2Me

C13H27

HN

OBn

C12H25

OPiv

O

O

BocHNBnO

BnOHO

O

OAcOAcOO

CO2Me

C13H27

HN

OAc

C12H25

OPiv

O

O

BocHNAcO

AcOO

O

OAcAcO

AcO

O

OAc

AcOAcO

AcO

O

OH

O

N3

BnOBnO

TBDPSO O

OBnOBnO

HOMeO2C

C13H27

HN

OBn

C12H25

OPiv

O

1821

a b,c

22 23

24 25

26 27

28

d

SPhO

OBnBnO

BnO

O

OAc

AcOAcO

AcO

O27

O

OBnOBnOO

CO2Me

C13H27

HN

OBn

C12H25

OPiv

O

O

BocHNBnO

BnOHO

23

f,g,h iGSL-4

e

+

+

+

Scheme 2 Preparation of GSL-4. The reagents were as follows (yields in parentheses). (a) Diphenylsulfoxide, Tf2O, tri-t-butylpyrimidine, CH2CH2 (64%).

(b) H2S, pyridine, Et3N; di-t-butyldicarbonate (82%). (c) HF-pyridine, THF (89%). (d) TMSOTf, 4 A MS, CH2Cl2 (94%). (e) MeONa, MeOH, Ac2O, DMAP,

pyridine (94%). (f) NBS, acetone, H2O (77%). (g) Diphenylsulfoxide, Tf2O, tri-t-butylpyrimidine, 3 A MS, CH2Cl2 (82%). (h) Pd/C, H2, MeOH, THF;

Ac2O, DMAP, pyridine (55%). (i) MeONa, MeOH, H2O, THF; trifluoroacetic acid, CH2Cl2 (66%). Ac, acetyl; Bn, benzyl; Boc, t-butyloxycarbonyl; Bz, benzoyl;

Piv, pivaloyl; TBDPS, t-butyldiphenylsilyl.

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NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007 561

resonances, the proton NMR spectra of isolated and synthetic GSL-4were indistinguishable (within the error range of the spectrometer;Supplementary Fig. 1 online). Particular attention was paid to reso-nances from the protons on anomeric carbons, because the chemicalshifts of these resonances are characteristic of the configurationand identity of the sugar. The carbon spectra of isolated and syntheticGSL-4 were indistinguishable, and TLC retention factors were identical.

Preparation of the GSL-KRN7000 hybrids a�GlcNH2-(1-4)-a-GalCer and a-GlcNAc-(1-4)-a-GalCer (Fig. 1) followed a pathwaysimilar to that giving GSL-2 (Scheme 3). That is, an appropriatelyprotected form of a-GalCer was generated (32) and coupled with theglucosamine derivative 19 (Scheme 1), yielding 33. Deprotection andreduction gave a-GlcNH2-(1-4)-a-GalCer. Peracylation, followed byester hydrolysis, gave a-GlcNAc-(1-4)-a-GalCer.

The collection of synthetic GSLs, isolated GSL-4 and GSL-KRN7000hybrids allowed observation of both the NKT cell–stimulatory proper-ties of these glycolipids and requirements for lysosomal glycolipidprocessing. NKT cell stimulation was observed by using a mousehybridoma, a human NKT cell line, splenocytes from B6 mice andNKT cells isolated from human plasma7,23. In each of these experi-ments, APCs were present; thus, not only were the glycolipidspresented by these cells, but they were also exposed to the glycosidasespresent in the lysosomes of these cells.

The stimulatory properties of the GSLs were compared to those ofPBS57 (34), a surrogate for KRN7000 (ref. 24), by using a mouse NKTcell hybridoma (DN32.D3) (Fig. 2a). In initial experiments with GSL-4isolated from S. paucimobilis, low stimulation of cytokine productionwas observed at relatively high concentrations of the glycolipid(B100 ng/ml). Careful purification, however, yielded a compoundthat stimulated only at higher concentrations. The stimulatory propertiesof GSL-1 would make a sample of GSL-4 contaminated with even a traceamount of GSL-1 appear to be stimulatory. Notably, synthetic GSL-4,GSL-3 and GSL-2 did not stimulate the mouse NKT cell hybridoma (datafor GSL-2 and GSL-3 not shown). Macrophages and dendritic cells wereused to verify that the result was not APC dependent. Similarly, onlyGSL-1 stimulated human NKT cells (Fig. 2b) when dendritic cells andperipheral blood lymphocytes (PBLs) were used as APCs. In the experi-ments with the human NKT cell line, we used PBS57 and iGb3 (ref. 15;35) as references. Modest stimulation was observed with GSL-1 and iGb3when dendritic cells were used as APCs. With PBLs as APCs, GSL-1 wasnearly as potent as PBS57. With splenocytes from B6 mice and with NKTcells isolated from human plasma, a similar result was obtained: PBS57

and GSL-1 caused both interferon-g (IFN-g)and interleukin 4 (IL-4) release, but GSL-3 andGSL-4 gave no response (Supplementary Figs.2 and 3 online).

Many different types of lysosomal glycosi-dases are present in APCs; consequently, it issurprising that GSL-2 is not truncated toGSL-1, yielding a stimulatory glycolipid.Similarly, GSL-3 and GSL-4 might also beexpected to be truncated to GSL-1. a-Man-nosidase25 and a-glycosidase A26 are knownto be active in lysosomes; thus, both GSL-3and GSL-4 should be truncated to GSL-2. Inhigher organisms, amino sugars are generallyacylated at nitrogen, and the glucosamines inGSL-2, GSL-3 and GSL-4 are non-acylated.We therefore reasoned that the glycosidasenecessary to cleave glucosamine from GSL-2might be absent or inactive in lysosomes. In

addition, lysosomes contain an acyltransfer enzyme that acylates theamines of the terminal a-glucosamine in heparin27. It is not clear whythe amine group in GSL-2 (and GSL-3 and GSL-4) is not acylated andsubsequently truncated to GSL-1. Lysosomal trafficking and transportof glycosylceramides is highly dependent on lipid transportproteins28,29, and it is possible that, while bound to lipid transferproteins, GSL-2 is not an effective substrate for acyltransfer enzymes.

To verify that glucosamine is not acylated and cleaved fromglycosylceramides in lysosomes, we examined the stimulatoryproperties of a-GlcNH2-(1-4)-a-GalCer (Fig. 1). Because we haveshown that other, similarly (1-4)-substituted, diglycosylceramides(that is, a-Gal-(1-4)-a-GalCer and b-GalNAc-(1-4)-a-GalCer)15 aretruncated to a-GalCer and stimulate NKT cells, lack of stimulation bya-GlcNH2-(1-4)-a-GalCer would provide evidence of the inability ofthe APC to acylate and cleave glucosamine from GSL-2. As a control,we prepared a-GlcNAc-(1-4)-a-GalCer (Fig. 1), which would be theproduct of acylation of a-GlcNH2-(1-4)-a-GalCer. a-N-Acetylgluco-saminidase is a lysosomal enzyme (whose absence results in Sanfilipposyndrome)30, which should cleave the N-acetylglucosamine froma-GlcNAc-(1-4)-a-GalCer to yield a-GalCer. Comparison of theNKT cell stimulatory properties of a-GlcNH2-(1-4)-a-GalCer anda-GlcNAc-(1-4)-a-GalCer demonstrated that a-GlcNH2-(1-4)-a-GalCer is non-stimulatory, whereas a-GlcNAc-(1-4)-a-GalCer is

1,000a b

1,0001,2001,4001,6001,8002,0002,200

900

800

700

600

500

400

300

200

100

0

800600400200

00.01 0.1 1 10

IL-2

(pg

ml–1

)

Glycolipid (ng ml–1) Glycolipid (ng ml–1)

IFN

-γ (

pg m

l–1)

100 1,000 0.1 1 10 100 1,000

Figure 2 Only GSL-1 and PBS57 cause significant stimulation of NKT cells. Two types of NKT cell were

stimulated with the following glycolipids: GSL-1, squares; GSL-4 (synthetic), circles; GSL-4 (isolated),

triangles; PBS57, diamonds; iGb3, crosses. (a) DN32.D3 cells (mouse NKT cell hybridomas). Filled

symbols, dendritic cells as APCs; open symbols, macrophages as APCs. (b) Human NKT cells. Filled

symbols, dendritic cells as APCs; open symbols, PBLs as APCs. Experiments were run in triplicate;

error bars represent 1 s.d.

PBS572,500

2,000

1,500

1,000

500

00.01 0.1 1 10

Glycolipid (ng ml–1)

IL-2

(pg

ml–1

)

100 1,000

α-GlcNH2-(1,4)-α-GalCer

α-GlcNAc-(1,4)-α-GalCer

Figure 3 GlcNAc from a-GlcNAc-(1-4)-a-GalCer is truncated by lysomal

processing to generate a stimulatory glycolipid. DN32.D3 cells were

stimulated with the indicated glycolipids by using dendritic cells as APCs.

Experiments were run in triplicate; error bars represent 1 s.d.

L E T TERS

562 VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY

stimulatory, albeit less than PBS57 (Fig. 3). This result supports theconclusion that glycolipids containing glucosamine are inefficientsubstrates for the lysosomal acyltransfer enzyme.

That GSL-1 is a potent stimulator of cytokine production by NKTcells and the other GSLs are non-stimulatory suggests that synthesis ofthese higher-order GSLs might be a mechanism of immune evasion.We have not determined the influence of stress on GSL1/GSL4 ratios,but this ratio might be exploited by bacteria to avoid detection.

The CD1d-NKT cell system provides a mechanism for surveillancefor bacterial glycolipids. An understanding of the glycolipids recog-nized by this system is essential for identification of the types oforganism that stimulate immune responses and the potential con-tributions of these organisms to immune disorders. Among bacterialglycolipids shown to stimulate NKT cells directly, those from theSphingomonadaceae family are the most potent. The GSLs includerelatively complex oligoglycosylceramides, and the synthesis of thisseries of glycolipids has provided both confirmation of their structureand the opportunity to determine stimulatory properties with homo-geneous materials. Our observation that, among the GSLs, only GSL-1is a potent stimulator suggests that the oligosaccharides in the higherGSLs are not directly presented by CD1d and recognized by NKT cells.Our studies with the GSL-KRN7000 hybrids demonstrated that thehigher GSLs are not truncated to GSL-1, because GSL-2 is not asubstrate for acyltransfer enzymes in lysosomes in APCs. Takentogether, these results define the extent to which NKT cells respondto different GSLs and the reason why higher-order GSLs are notconverted to GSL-1 in lysosomes. These results not only add to ourunderstanding of NKT cell responses but also may lead to improvedantigens for these immunoregulatory cells.

METHODSDetailed descriptions of the preparation of GSL1-GSL4 and GSL-KRN7000

hybrids, NMR spectra of final compounds (Supplementary Fig. 4 online) and

an LC chromatogram of synthetic GSL4 (Supplementary Fig. 5 online) are

provided in the Supplementary Methods online.

Stimulation with cell lines. For observation of NKT cell stimulation with cell

lines, 100,000 cells of a DN32.D3 hybridoma or 250,000 cells of a human NKT

cell line29,30 were incubated with the indicated glycolipid in the presence of the

specified APCs (100,000 for mouse cell assays and 250,000 for human cell

assays). Human and mouse dendritic cells were prepared as described8. Bone

marrow–derived mouse macrophages were collected after 6 d of culture in RPMI

medium containing 10% fetal calf serum (FCS) with 2 ng/ml of recombinant

mouse macrophage colony-stimulating factor (R&D systems). Cell culture

supernatants were assayed after 24 h for IL-2 release (DN32.D3 hybridoma

stimulation) or 48 h for IFN-g production (human NKT cell line) by ELISA

(R&D Systems). Each experiment was performed a minimum of three times.

Stimulation with primary cultures. For stimulation experiments with NKT

cells isolated from human plasma, PBLs were obtained after Ficoll centrifuga-

tion of heparinized blood. PBLs were incubated with KRN7000 (100 ng/ml) and

IL-2 (100 U/ml) for 10 d in medium containing 10% AB serum. NKT cells were

sorted as CD1d-KRN7000+Va24+ cells from human PBLs of two different

donors. For stimulation experiments, the NKT cell lines were expanded with

irradiated Epstein-Barr virus cells and PBLs in the presence of 1 mg/ml of

phytohemagglutinin and 100 U/ml of IL-2 in 10% AB serum for 2–3 weeks until

reaching an exponential growth phase. The cells were then washed and 250,000

NKT cells in coculture with 250,000 irradiated monocyte-derived dendritic cells

(peripheral blood mononuclear cells cultured for 5 d in the presence of 100 mg/

ml each of granulocyte-macrophage CSF and IL-4 (R&D Systems) as described)

were incubated with the indicated concentrations of glycolipid in 96-well

round-bottom plates containing 250 ml of 10% AB serum per well. For

stimulation experiments with B6 mouse splenocytes, spleen cell suspensions

(5 � 105 cells/well) were exposed to the indicated concentrations of glycolipids

in 96-well round-bottom plates in RPMI 1640 (Biofluids) supplemented with

glutamine, antibiotics, 5 � 10�5 M b-mercaptoethanol and 10% FCS.

Cytokine quantification. Cytokine concentrations were determined from

cell culture supernatants, which were assayed at 24 h for IL-2 release

(DN32.D3 hybridoma stimulation) or 48 h later for IFN-g production

(human NKT cell line or splenocytes) by ELISA (R&D Systems; lower detection

limit of 15 pg/ml).

Note: Supplementary information and chemical compound information is available onthe Nature Chemical Biology website.

ACKNOWLEDGMENTSWe acknowledge financial support from the National Institutes of Health (NIAIDAI053725). J.M. is a Cancer Research Institute fellow and was supported by agrant from the Lupus Research Institute. A.B. is a Howard Hughes MedicalInstitute Investigator.

AUTHOR CONTRIBUTIONSX.L., S.D., Z.Z. and R.D.G. synthesized glycolipids; J.M. and D.Z determined NKTcell stimulatory activity of glycolipids; N.M. isolated glycolipids; L.T., A.B. andP.B.S. designed the project.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturechemicalbiology

Reprints and permissions information is available online at http://npg.nature.com/

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